Abstract

The global transcriptional coactivators CREB-binding protein (CBP) and the closely related p300 interact with over 312 proteins, making them among the most heavily connected hubs in the known mammalian protein-protein interactome. It is largely uncertain, however, if these interactions are important in specific cell lineages of adult animals, as homozygous null mutations in either CBP or p300 result in early embryonic lethality in mice. Here we describe a Cre/LoxP conditional p300 null allele (p300flox) that allows for the temporal and tissue-specific inactivation of p300. We used mice carrying p300flox and a CBP conditional knockout allele (CBPflox) in conjunction with an Lck-Cre transgene to delete CBP and p300 starting at the CD4- CD8- double-negative thymocyte stage of T-cell development. Loss of either p300 or CBP led to a decrease in CD4+ CD8+ double-positive thymocytes, but an increase in the percentage of CD8+ single-positive thymocytes seen in CBP mutant mice was not observed in p300 mutants. T cells completely lacking both CBP and p300 did not develop normally and were nonexistent or very rare in the periphery, however. T cells lacking CBP or p300 had reduced tumor necrosis factor alpha gene expression in response to phorbol ester and ionophore, while signal-responsive gene expression in CBP- or p300-deficient macrophages was largely intact. Thus, CBP and p300 each supply a surprising degree of redundant coactivation capacity in T cells and macrophages, although each gene has also unique properties in thymocyte development.

The CBP and p300 interactome: 312 viral and mammalian proteins that interact physically or functionally with CBP or p300 in vitro. One hundred ninety-six proteins that are encoded by essential genes in mice (i.e., mutation results in a phenotype) are indicated in boldface type, including 56 genes that are essential for T cells (indicated by an asterisk). The source for phenotype information is Mouse Genome Informatics (http://www.informatics.jax.org/). For more detailed and up to date information on the interactome, see http://www.stjude.org/brindle. References are indicated in parentheses.

Ablation of p300 and CBP protein in mutant thymocytes. (a) Western blot of control and mutant thymocyte whole-cell extracts (indicated) using C-terminal antibodies (α) to p300 (top panel) and CBP (middle panel) and an antibody against CREB (lower panel). (b) Single-cell suspensions of thymuses from control (p300flox/flox) (upper panels), p300flox/flox; Lck-Cre (middle), and CBPflox/flox; Lck-Cre (lower) mice were stained with 4′,6′-diaminido-2-phenylindole (DAPI) to visualize nuclei and antibodies specific for the C termini of p300 or CBP (indicated). Detection was by anti-rabbit immunoglobulin G conjugated with fluorescein isothiocyanate.

Moderate to strong selection against peripheral T cells lacking three functional alleles of CBP and p300. Percent deletion of CBPflox and p300flox alleles in the thymus and affinity-purified T cells from the spleen and lymph node (LN) of CBPflox/flox; p300+/flox; Lck-Cre (a and c) or CBP+/flox; p300flox/flox; Lck-Cre (b) mice. (c) Deletion frequency in the thymus does not directly impinge on the deletion percentage in the periphery (compare panels b and c). Deletion percentages for CBPflox and p300flox were determined by semiquantitative PCR of genomic DNA corrected for nonlinearity by use of a standard curve made of samples with deletion frequencies determined by quantitative Southern blot.

Examination of endogenous TCR-signaling-responsive gene expression in splenic T cells lacking p300 or CBP. (a and b) Splenocytes from mice bearing Lck-Cre and Z/EG reporter transgenes were sorted for GFP-expressing control (Lck-Cre; Z/EG), p300 null (p300flox/flox; Lck-Cre; Z/EG), or CBP null (p300flox/flox; Lck-Cre; Z/EG) splenic T cells. Cells were treated ex vivo with 1 ng/ml PMA and 200 ng/ml calcium ionophore (Iono) for 3 h, and qRT-PCR was performed to determine mRNA expression of IL-2 (a) and TNF-α (b). Expression was normalized to GAPDH expression, and treated control cells were arbitrarily set to 100 in each experiment. Graphs represent the average results from two independent experiments using cells from two different mice of each genotype (n = 2).

Examination of endogenous gene expression in BMDMs deficient for either CBP or p300. (a) lysMcre-mediated loss of CBP in BMDMs (left panel). Nuclear extracts from BMDMs cultured ex vivo from CBPflox/flox or two different CBPflox/flox; lysMcre mice showed CBP deficiency as measured by immunoblotting with CBP-specific antisera. p300 levels were unaffected. (Right panel) p300 deletion in BMDMs was confirmed by semiquantitative PCR of genomic DNA. (b) Normal CBP levels are not required for the TLR4-mediated induction of TNF-α expression. Northern blot analysis of TNF-α mRNA induction in response to stimulation through the TLR4 pathway. BMDMs were plated at 2 × 106 cells per well and stimulated with Escherichia coli LPS at 100 ng/ml for the times indicated. RNA was isolated and probed with a TNF-α cDNA probe. Ethidium bromide-stained rRNA is the loading control (bottom panels). (c) CBP is not required for the IL-4-mediated induction of arginase 1 (Arg1) expression. BMDMs were stimulated with IL-4 (10 ng/ml) or left unstimulated (−) for the times shown, and lysates were analyzed for Arg1 expression by immunoblotting. The nonspecific band serves as a loading control (asterisk). (d) Normal p300 levels are not required for the TLR4-mediated induction of TNF-α expression. Total RNA was isolated and analyzed as shown in panel b. (e) Normal p300 levels are not required for the IL-4-mediated induction of Arg1 expression. BMDMs were plated and stimulated with IL-4 as shown in panel c. (f) Normal p300 levels are not required for the IFN-γ-mediated induction of IRF-1 expression but may be required for the normal kinetics of iNOS production. BMDMs were stimulated with LPS and IFN-γ to induce the expression of IRF-1 and iNOS, and lysates were analyzed by immunoblotting. The nonspecific band serves as a loading control (asterisk). Note that although IRF-1 levels are normal in p300flox/flox; lysMcre BMDMs compared to control cells, the induction of iNOS expression is slightly delayed at the 6-h time point but eventually reaches robust levels.